A Practical Guide to Containment: Greenhouse Research with Transgenic Plants and Microbes

Section III. Biosafety Levels

THE PURPOSE OF CONTAINMENT IS TO PREVENT the transfer of recombinant DNA from transgenic organisms inside the greenhouse to populations outside the greenhouse. Section III of the NIH Guidelines describes four physical containment levels for experiments involving recombinant DNA molecules. It further categorizes experiments according to specific risk criteria and assigns them to one of the four biosafety levels, BL1-P through BL4-P.

Appendix P of the Guidelines specifies the physical and biological containment conditions and practices required for greenhouse experiments for each biosafety level. A brief description of the four biosafety levels and the criteria used by the NIH Guidelines for assigning experiments to each category are provided here. It is the responsibility of the IBC and PI to determine the appropriate biosafety level. When making a biosafety level assignment, consider the following criteria:

Source and nature of the introduced DNA: whether from an exotic infectious agent or pathogenic organism; and whether a fragment of DNA or complete genome;

Recipient organism: mode and ease of dissemination; invasiveness; whether a noxious weed or capable of interbreeding with noxious weeds; potential for outcrossing between recipient organisms and nearby related species; and potential for detrimental impact on natural or managed ecosystems;

Nature of expressed protein: whether a vertebrate toxin or potential or known allergen; and whether toxic to other organisms in local environment;

Local environment: nature and importance of nearby crops; presence of sexually compatible wild or weedy species; and

Experimental procedures: transfer to or from greenhouse; and necessary containment measures.

Sound scientific principles and a thorough knowledge of the recipient organism and its mode of dissemination are the basis for designating a suitable level of containment. A brief comparison of criteria used in the Guidelines to assign an appropriate biosafety level is shown in Table 2. The table shows that as the potential risk to the environment increases, increasingly stringent requirements for containment are indicated. When applicable, physical containment requirements may be eased by the addition of measures for biological containment, indicated by the "+" sign. (Biological containment is described in Section IV, Elements of Containment.)

CRITERIA	TRANSGENIC PLANTS	TRANGENIC MICROBES		TRANSGENIC INSECTS/ANIMALS/ ASSOC. MICROBES
		Exotic	Non-Exotic
Not a noxious weed or cannot outcross with one	BL1-P
Not easily disseminated			BL1-P
No detriment to environment		BL2-P or BL1-P +	BL1-P	BL2-P or BL1-P +
Noxious weed or can interbreed with weeds	BL2-P or BL1-P +
Contains complete genome of non-EIA*	BL2-P or BL1-P +
Contains genome of EIA	BL3-P or BL2-P +
Treated with an EIA	BL3-P or BL2-P +
Detriment to environment		BL3-P-4**	BL2-P or BL1-P +	BL3-P or BL2-P +
Involves EIA with detriment to environment	BL3-P or BL2-P +
May reconstitute genome of infectious agent in planta	BL3-P or BL2-P +
Contains Vertebrate Toxin	BL3-P	BL3-P	BL3-P

*EIA — Exotic Infectious Agent
**BL4-P containment is recommended only for experiments with readily transmissible exotic infectious agents whether transgenic or not, such as air-borne fungi or viruses in the presence of their arthropod vectors that have the potential of being serious pathogens of major US crops.

Experiments that Are Exempt

Experiments that do not present a risk to health or the environment are exempt from the NIH Guidelines and do not require the approval of the local IBC. For example, research using synthetic DNA molecules that are not part of any organism or virus, or research using only DNA segments from a single nonchromosomal or viral source, are exempt. Also exempt are experiments in which the DNA from a particular host organism is propagated only in that same organism, as would be the case for research designed to splice DNA segments taken from wheat into the genome of the same or another wheat variety. This exemption applies to DNA segments regardless of whether they were obtained from the host chromosomes, chloroplasts, mitochondria or plasmids, as long as the fragment is propagated only in that same host, and that no other DNA is used, including promoters and enhancers. Finally, the Guidelines exempt research involving the transfer of DNA between two different species if they are known to exchange DNA by well established physiological means. Appendix A of the NIH Guidelines contains a periodically revised list of these natural exchangers ¹⁰. Currently, most organisms on this list are bacteria and yeast species, but some genera of plant pathogenic bacteria are included.

Biosafety Level 1 for Plants (BL1-P)

The BL1-P designation provides for a low level of containment for experiments involving transgenic plants in which there is no evidence that the modified organism would be able to survive and spread in the environment and, if accidentally released, would not pose an environmental risk. For example, an experiment designed to study transgenic potato plants containing cloned genes for insect resistance obtained from primitive potato cultivars would be classified as BL1-P.

BL1-P also applies to DNA-modified common microorganisms that cannot spread rapidly and are not known to have any negative effects on either natural or managed ecosystems, such as Rhizobium and Agrobacterium. A BL1-P designation would be assigned, for example, to an experiment that uses a transgenic strain of Rhizobium containing Agrobacterium genes known to affect root colonization, or plants using Agrobacterium DNA segments as part of the transformation process.

Biosafety Level 2 for Plants (BL2-P)

BL2-P is assigned to experiments with transgenic plants and associated organisms, which, if released outside the greenhouse, could be viable in the surrounding environment but would have a negligible impact or could be readily managed. BL2-P is required for transgenic plants that may exhibit a new weedy characteristic or that may be capable of interbreeding with weeds or related species growing in the vicinity. For example, greenhouse tests of transgenic sunflower containing wheat genes intended to confer resistance to the fungus

Sclerotinia would be classified BL2-P because sunflower is capable both of hybridizing with wild relatives, and becoming established as a volunteer weed.

BL2-P containment is assigned to transgenic experiments that use the entire genome of an indigenous infectious agent or pathogen. This level of containment is also appropriate for transgenic plant-associated microorganisms that are either indigenous to the area and potentially harmful to the environment but manageable, or are exotic but have no potential for causing serious harm to managed or natural ecosystems. The BL2-P classification likewise applies to experiments using plant-associated transgenic insects or small animals as long as they pose no threat to managed or natural ecosystems.

Biosafety Level 3 for Plants (BL3-P)

BL3-P facilities are designed to prevent the accidental release of transgenic plants, plant pathogens, or other organisms that have a recognized potential for significant detrimental impact on the environment. This category also applies to non-GMO plant research that involves exotic infectious agents capable of causing serious environmental harm. In these cases, it is the pest or pathogen that requires containment; the transgenic plant itself may pose no threat. BL3-P is also recommended for transgenic plants containing genes from an exotic infectious agent in which a complete functional genome of the infectious agent could possibly be reconstituted. Experiments using transgenic plants or organisms that contain genes coding for vertebrate toxins are likewise conducted at BL3-P. Lastly, BL3-P is recommended for experiments using transgenic microbial pathogens of insects or small animals that associate with plants, if the pathogen has the potential to cause harm to the local environment.

Examples of research requiring BL3-P facilities:

Testing citrus plants engineered to be resistant to Asiatic Bacterial Canker by infecting them with the disease pathogen, which, if released in Florida, could devastate the commercial citrus crop;

Inoculating transgenic peanut plants containing fungal resistance genes with Aspergillus flavus, the organism responsible for producing the potent vertebrate mycotoxin, aflatoxin.

Biosafety Level 4 for Plants (BL4-P)

Cicadulina spp., in challenge inoculations. This devastating virus is not found in the United States, however leafhopper species capable of transmitting it are present. Thus the experiment using both virus and vector poses a significant risk should either escape the containment facility; in this case, the transgenic maize plant does not itself pose a risk.

¹⁰ http://www4.od.nih.gov/oba/RAC/guidelines/appendix_a.htm